We have developed prototype instrumentation enabling in vivo EPR spectroscopy and high resolution spatial or spectral-spatial imaging of free radicals and oxygen. This instrumentation has been applied to provide important information regarding the spatial location of free radicals and paramagnetic molecules in ex vivo and in vivo biological systems. The prototype 1-2 GHz instrument has enabled measurement and imaging of oxygen, free radical compounds, and drugs in living tissues or animals (small mice) up to 25 mm size with submillimeter resolution. The feasibility of in vivo imaging of nitric oxide generation was demonstrated. This new technology has great potential biomedical importance, however, several factors presently limit broad application. These include the need to: accommodate larger ex vivo and in vivo samples; eliminate gradient induced perturbations of static magnetic field control; enhance sensitivity and limit motion induced noise; increase the speed of image acquisition; and develop a common user interface/software to enable rapid setup and application with transition from routine spectroscopy to 2D, 3D, or 4D spatial or spectral-spatial imaging. These critical needs are to be addressed with a combination of system hardware and software development as described in five projects: I. Development of a new larger gap magnet and gradient assembly with optimized hall probe arrangement; II. Microwave bridge development for 750-800 MHz with narrow band frequency designs; III. Optimized resonator design for a range of user applications with automatic tuning and automatic coupling arrangements; IV. Development and integration of hardware and software to increase the speed and efficiency of image acquisition; V. Development of a user software interface enabling the application scientist to perform routine experiments and analyze the spectral and image data obtained in real time. These innovations will provide solutions to the problems which have limited the broad biomedical application of this promising technology. This proposal will lead to the creation of a resource center with two EPR imaging instruments, each optimized for important biomedical applications.